The ultimate goal of this research proposal is to develop an injectable cell-polymer composite that will aid in the repair of tissue in osteochondral defects. Based on a novel hydrogel material developed in our laboratory, oligo(poly(ethylene glycol) fumarate), these constructs will be photocrosslinked in situ in the presence of mesenchymal stem cells (MSCs). A three-step approach will be followed to engineer optimal injectable constructs using a combination of cells, biodegradable scaffolds, and spatially and temporally graded release of a growth factor. The first step will be to determine how hydrogel mesh size (distance between crosslinks) affects differentiation of embedded MSCs in vitro and in a rabbit osteochondral defect model. The hydrogel system will then be modified through the synthesis of crosslinking molecules with peptide sequences that are degradable by matrix metalloproteinases found in articular cartilage. The optimal initial mesh size, determined previously, will be used as the basis for study of the effect of different crosslinker concentrations on degradation of the hydrogel due to differentiation of embedded MSCs in vitro and in vivo. Finally, a bilayered construct will be created from the biodegradable hydrogel with the optimal crosslinker concentration in which a gradient of TGF-beta1, released in a controlled manner in response to enzymes produced as neotissue forms, is established to promote cartilage formation in the top half of the construct and bone formation in the lower half. Efficacy of tissue formation from this construct will be tested in vitro and in vivo. The proposed project will provide clinically valuable information regarding new composite constructs for improved repair of osteochondral defects, thus providing a method to generate cartilage repair tissue, which will not degenerate over time, a major limitation with current techniques.